石山苣苔屬（苦苣苔科）花形態(tài)演化及分類學(xué)意義

盧永彬　黃俞淞　許為斌　黃潔　劉演　向春雷　張強(qiáng)

摘要： 石山苣苔屬（苦苣苔科）約30種，主要分布于我國南部的石灰?guī)r地區(qū)。目前該屬已知物種數(shù)雖少但花形態(tài)極其多樣，是該科中分類最為困難的類群之一?；诜肿幼C據(jù)，其它8個(gè)屬中花形態(tài)迥異的一些物種被并入石山苣苔屬。然而，該屬花形態(tài)的演化趨勢缺乏系統(tǒng)性的研究，傳統(tǒng)分類對(duì)屬的界定與分子系統(tǒng)學(xué)研究結(jié)果相矛盾的原因，以及是否有形態(tài)特征支持新界定的石山苣苔屬還不清楚。該研究中，總共編碼了19種石山苣苔屬植物和9種報(bào)春苣苔屬植物的35個(gè)形態(tài)特征，其中包括26個(gè)花部形態(tài)特征，在分子系統(tǒng)樹上追蹤了它們的演化路徑。結(jié)果表明：無論屬內(nèi)還是屬間，多數(shù)花部形態(tài)特征，尤其以往屬的分類界定特征，在演化過程中變化頻繁且發(fā)生了高度同塑性演化，這是導(dǎo)致傳統(tǒng)形態(tài)分類不自然的關(guān)鍵因素。此外，在觀察研究的所有特征中，花絲和柱頭的差異可能在石山苣苔屬植物共同祖先中經(jīng)歷了演變，或可用于區(qū)分石山苣苔屬與其姐妹報(bào)春苣苔屬的大多數(shù)種類。因此，在苦苣苔科植物的分類學(xué)研究中應(yīng)當(dāng)慎用這些花部性狀作為分類依據(jù)，而且應(yīng)對(duì)形態(tài)特征進(jìn)行廣泛地觀察研究，在密集的取樣和分辨率更高、更可靠的系統(tǒng)樹上追蹤它們的演化規(guī)律。更為重要的是，需要進(jìn)一步研究導(dǎo)致復(fù)雜形態(tài)性狀演化的內(nèi)在分子調(diào)控機(jī)理和外在的自然選擇動(dòng)力，最終更加深入地理解石山苣苔屬等典型喀斯特植物的演化過程和機(jī)理。

關(guān)鍵詞： 分類， 花部特征， 苦苣苔科， 同塑性演化， 喀斯特植物， 石山苣苔屬

Petrocodon Hance （Gesneriaceae）， firstly established in 1883， remained as a monotypic genus （only containing Petrocodon dealbatus） over a century. It is perennial herb and has campanulate corolla with indistinctly twolipped limb， being different from most other Gesneriaceae， which usually possess distinctly twolipped limb with lobes different in size and/or shape. The species is distributed in South and Central China， mostly growing in the crevices of limestone rocks. No more species of the genus were reported in the following century. Till the last decade， several new species of Petrocodon were finally discovered and described from South China （Wei， 2007； Wei et al， 2010）.

The phylogenetic position and the boundary between Petrocodon s.s. and other closelyrelated genera were revealed by the recent molecular phylogenetic studies （Weber et al， 2011b； Xu et al， 2014）. The monotypic genera （Calcareoboea C. Y. Wu ex H. W. Li， Paralagarosolen Wei， Dolicholoma D. Fang & W. T. Wang and Tengia Chun） and one to a few species from Wentsaiboea Fang & Qin， Primulina Hance， Lagarosolen Wang and Didymocarpus Wallich were included into Petrocodon and the expanded genus was indicated to be most close to Primulina according to the molecular phylogenetic studies based on the chloroplast trnLtrnF and nuclear ribosome ITS DNA sequences （Weber et al， 2011b； Xu et al， 2014）. The redefinition as well as more new species recently published enlarged Petrocodon to contain ca. 30 species （Weber et al， 2011b； Wen et al， 2012； Chen et al， 2014； Hong et al， 2014； Xu et al， 2014； Yu et al， 2015； Guo et al， 2016； Lu et al， 2017）. It is more noteworthy is that the expanded genus represent one of the most varied genera in the Old World， comparable to those in the New World （Weber et al， 2011b）.

The morphological traits of the expanded Petrocodon， particularly those of floral parts， are remarkably diverse among the lineages of Old World Gesneriaceae. The floral variation of Petrocodon covers almost all aspects of the floral characters. For example， the symmetry of corolla includes zygomorphy and almost actinomorphy； most species have corolla limb with two adaxial （dorsal） lobes and three abaxial （ventral） lobes but two species of the genus have corolla limb with a very unusual pattern of four adaxial lobes and one abaxial lobe [corresponding to the terminology of fourtoothed upper lip and onetoothed lower lip according to Weber et al （2011b）]； corolla shape is variable from campanulate， tubular， funnelform， urceolate to salverform； corolla coloration is diverse， including pure white， purple， yellow and bright red； stamen numbers per flower are usually two but Pet. scopulorum （previous Tengia scopulorum） and one recently published species Pet. hunanensis （Yu et al， 2015） belonging to the genus possess five and four fertile stamens， respectively. The general appearance of the floral diversity of Petrocodon is as shown in Fig. 1.

The redefinition of Petrocodon and the inclusion of species from other eight genera， could imply that morphological characters， particularly those diagnostic traits the traditional taxonomy was based on， might experience complicated evolution such as homoplasy （i.e. convergence， parallel or reversal evolution）， dramatic morphological divergence of closely related species， and/or plesiomorphy remained in different lineages etc. However， little is known about the evolutionary trends of the diverse morphological characters in the expanded genus and their taxonomic significance， though the previous molecular phylogenetic study sampled the majority of the included species （Weber et al， 2011b； Chen et al， 2014）. It remains unknown whether there is any floral or vegetative characters that can differentiate Petrocodon from its sister genus Primulina and other close relatives， and characterize each of the lineages in the molecular phylogenetic tree within the genus. To explore these issues needs carefully examination of the diverse morphological characters of Petrocodon， comparing with other close relatives， and tracing their evolution based on a robust phylogenetic framework suggested by molecular phylogenetic studies.

In this study， we examined and coded 35 morphological characters （including 26 flora traits） of Petrocodon and some outgroup taxa of Primulina， traced the evolutionary trends of these characters on the phylogenetic tree reconstructed based on DNA sequences downloaded from the public accessible database of National Centre of Biological Information （NCBI）. The aims we targeted to explore included： （1） the evolutionary trends how the diverse morphologies evolved； （2） the potential synapomorphy that can characterize and differentiate Petrocodon from its close relatives. We also discuss the implications of the inferred morphological evolution for future classification of Gesneriaceae.

1Materials and Methods

1.1 Materials examined

A total of 35 morphological characters （including 26 floral characters） of 19 Petrocodon species and 9 outgroup species of Primulina were examined and coded numerically. The morphological characters were coded and validated according to the original description of the species， the related monographs such as Flora of China （Wang et al， 1998）， Plants of Gesneriaceae in China （Li & Wang， 2005） and Gesneriaceae of South China （Wei et al， 2010）， and the observation of transplanted individuals and/or the field investigations.

We coded different states of the same character with different numerics. For those possessing mosaic character states， we coded them with all contained states. For example， for the corolla coloration， Pet. tiandengensis has corolla with pale blue lobes and white tube， we coded it with these two character states （0 and 2， representing bluish violet and white， respectively.） For the characters lacking of observation and record， we treated them as missing. All the coded traits are listed in Table 1.

1.2 Selection of DNA markers and sampling strategy strategy

As the plastid trnLtrnF intergenic spacer and nuclear ribosome internal transcribed spacer （ITS） sequences have been most frequently used for phylogenetic analysis in Gesneriaceae， particularly in the subfamily Didymocarpoideae including Petrocodon from the Old World （Denduangboripant et al， 2007； Wang et al， 2010； Mller et al， 2011a， b； Weber et al， 2011b； Chen et al， 2014）， these two DNA fragments were therefore selected and downloaded from GenBank for the present study.

As shown by some previous studies （Weber et al， 2011b； Chen et al， 2014）， the newly redefined genus Primulina is the closest relative to Petrocodon， therefore， all the trnLtrnF and ITS sequences of Petrocodon taxa and a few of Primulina representatives available from GenBank were included. The sequences of some other Chinese Gesneriaceae， namely Allocheilos W. T. Wang， and Oreocharis mileensis which also possess the rare corolla character of four adaxial lobes and one abaxial lobe （Fig. 2） were also included. Some other Old World Gesneriaceae， e.g. Deinocheilos W. T. Wang and Gyrocheilos W. T. Wang with another pattern of special corolla structure （unity of the adaxial lip with normal tripartition of the abaxial lip） as well as several other Oreocharis species were also downloaded and included in the present phylogenetic analyses. Finally， sequences of some representative species of Lysionotus， Cyrtandra and Streptocarpus were included and used as the more distant outgroups according to the previous largerscale phylogenetic analyses （Roalson & Roberts， 2016）. The trnLtrnF data included 60 accessions representing 45 species and the ITS data encompassed 54 accessions of 44 species. The GenBank accessions of all the downloaded sequences are as listed in Table S1 in supporting information.

1.3 Sequence assembling and phylogenetic analyses

All the sequences were aligned using the software MUSCLE 3.8.31 （Edgar， 2004）， and then manually adjusted using Bioedit 5.0.9 （Hall， 1999）. The flank regions which were ambiguously aligned or possessed numerous missing nucleotides for the majority of the accessions were deleted.

The maximum likelihood （ML） in RaxMLVIHPC （Stamatakis， 2006）， maximum parsimony （MP） in PAUP 4.10 （Swofford， 2002） and the Bayesian inference （BI） in MrBayes 3.2.6 （Ronquist et al， 2012） were employed to reconstruct the phylogenies. First， we conducted the ML analyses for each of the two regions under the settings of 1 000 rapid bootstrap searches and thereafter a thorough ML search with the substitution model GTR+G. The congruence of the phylogenetic signals of the two loci were assessed by comparing the two ML majorityrule consensus trees using a bootstrap support value of 70% as an arbitrary threshold， meaning that the nodes showing incongruent relationships between trnLtrnF and ITS ML majorityrule consensus trees with bootstrap support value above 70% were assumed to be strong phylogenetic conflict and against combined analysis of the two loci. For the ML analysis of the combined data， the same parameters were settled. For the MP analysis of the combined data， the MP settings were heuristic searches of 1 000 replicates of random sequence addition， tree bisection and reconnection （TBR） swapping and all MP trees saved at each replicate （Multree on）； for the bootstrap analysis， 1 000 bootstrap pseudoreplicates were conducted， each with 10 replicates of random sequence addition and a maximum of 5 000 trees saved for each bootstrap pseudoreplicate. For the BI analysis， 100 000 000 generations were run with four chains in two parallel runs and one tree every 5 000 generations were sampled with a burnin of the first 5 000 trees discarded. The convergence of the two parallel runs was guaranteed by the splitting frequency less than 0.005. All other parameters were set as default.

We reconstructed the ancestral floral morphologies and traced the evolutionary trends based on the molecular phylogenetic tree generated from the combined data with only Petrocodon and Primulina taxa preserved using Fitch maximum parsimony implemented in Mesquite 2.01 （Maddison & Maddison， 2007）. The phylogenetic tree to which the morphological characters were mapped was modified to comprise only one representative accession per species， with additional repetitive accessions of the same species pruned. All character states were treated as unordered， with all other settings left as default.

2Results and Analysis

2.1 Phylogenetic analysis of Petrocodon and its allies

The aligned trnLtrnF and ITS datasets were 896 and 744 base pairs including 74 and 286 informative sites， and 72 and 123 variable but parsimony uninformative sites， respectively. The combined matrix thus consisted of 1 640 characters including 555 variable sites with 360 informative sites. The parameters of consistency index （CI）， retention index （RI） and homoplasy index （HI） were 0.836， 0.904， and 0.164， and 0.553， 0.736 and 0.447 for the trnLtrnF and ITS data， respectively.

For the combined data， the ML， BI and MP analyses yielded 50% majorityrule consensus trees with congruent topologies （Fig. 3）. The monophyly of Petrocodon was recovered with moderate to high support values （BSML = 65%； PP= 0.98； BSMP = 100%）， and the genus was fully supported to be the sister of Primulina （BSML = 100%； PP= 1.00； BSMP = 100%）， which are in line with previous phylogenetic analyses （Weber et al， 2011b； Xu et al， 2014； Guo et al， 2016； Lu et al， 2017）. The relationships within Petrocodon are as shown in Fig. 3.

2.2 Floral evolution in Petrocodon

Mapping morphological characters to the molecular phylogeny clearly indicated most of the floral characters evolved frequently and highly homoplasiously in Petrocodon. The floral symmetry evolved twice in parallel from zygomorphy into almost actinomorphy （Fig. S1 in supporting information）； the ancestral corolla shape

Fig. 1Floral diversity of Petrocodon

A. Petrocodon tiandengensis； B. Pet. pseudocoriaceifolius； C. Pet. longgangensis； D. Pet. lui； E. Pet. guangxiensis； F. Pet. dealbatus； G. Pet. laxicymosus； H. Pet. coccineus； I. Pet. coriaceifolius； J. Pet. niveolanosus； K. Pet. hancei； L. Pet. hechiensis； M. Pet. jingxiensis； N. Pet. fangianus； O. Pet. jasminiflorus.

Fig. 2Parallel evolution of four adaxial corolla lobes and one abaxial lobe in separate Gesneriaceae lineages

A. Petrolodon retroflexus； B. Pet. coccineus； C. Oreocharis mileensis； D. Allocheilos cortusiflorus.

Fig. 3Maximum likelihood phylogenetic tree based on the combined trnLtrnF and ITS data， showing evolutionary relationships of Petrocodon and its allies

ML bootstrap support， BI posterior probability and MP bootstrap support values are listed to the corresponding nodes. * stands for the bootstrap support or posterior probability equal to 100% or 1.00； - stands for the bootstrap support or posterior probability below 50% or 0.50.

Fig. 4Evolutionary trends of four key representative floral characters， showing highly homoplasious evolution of these characters

Fig. 5Two species pairs which are morphologically similar with each other （especially for the floral characters） but are genetically distant

A-B. Primulina renifolia； C-D. Petrocodon tiandengensis； E. Primulina tabacum； F. Petrocodon guangxiensis.

was inferred to be tubular and evolved frequently （a total of 13 times of changes） among variable states， including twice convergently into campanulate and thrice into funnelform； the floral coloration was inferred to be purple ancestrally and experienced 13 times of changes， including six independent transformations in parallel into white； even the very unusual corolla structure of four adaxial lobes and one abaxial lobe evolved twice in parallel from the ancestral and common state of two adaxial lobes and three abaxial lobes in Petrocodon， with additional same two shifts in other Old World Gesneriaceae （Allocheilos and Oreocharis mileensis） also from southern China； the stigma also experienced eight changes， seven from bifid into unity （being solitary） in parallel and one reversal back from unity into bifid （Fig. 4）. Most other floral characters were also suggested to experience multiple homoplasious changes （data not shown）.

3Discussion

3.1 Frequent changes， convergent and parallel evolution of the previously assumed taxonomysignificant floral characters

Mapping the morphological characters to the phylogenetic tree clearly showed that most of the floral characters of Petrocodon experienced complicated evolution， i.e. frequent changes and multiple convergences and parallel changes， within and beyond the genus （Fig. 4）. This seems especially true for those diagnostic characters that were used to delimit the taxonomic units， which could have misled the traditional taxonomic treatments of the genera.

Floral （corolla） symmetry varied in Gesneriaceae and was assumed to be one of taxonomysignificant characters. Actinomorphic floral corolla symmetry， a relative rare state possessed only by a few Old World Gesneriaceae， was assumed to be a diagnostic character and mainly used to differentiate Primulina tabacum from other close relatives， e.g. the previous genus Chirita. Later， another morphologically similar new species Pri. guangxiensis which has general floral appearance and also almost actinomorphic corolla like Primulina tabacum was described and added to the monotypic genus （Liu et al， 2011）. However， the molecular phylogenetic analyses clearly indicated that Pri. tabacum was deeply embedded in the redefined genus Primulina while Pri. guangxiensis was nested within the redefined genus Petrocodon， strongly suggesting parallel evolution of floral symmetry from zygomorphy to actinomorphy. Some other Gesneriaceae in China （e.g. the previous Thamnocharis Wang， Bournea Oliv. and Conandron Sieb. & Zucc.） were also characterized by actinomorphic corolla， whereas， these similarities were suggested to be resulted from morphological convergence （factually parallel evolution） rather than common ancestry according to the molecular phylogenetic analyses （Wang et al， 2010）. Even in Petrocodon， the floral symmetry transformation from zygomorphy into almost actinomorphy was also suggested to have happen twice independently， leading to Petrocodon guangxiensis and Pet. scopulorum， respectively. All these suggest that the floral symmetry can evolve rapidly and in parallel and it is not suitable to be used as the diagnostic character to delimit taxonomic units （i.e. genus） in Gesneriaceae.

The corolla shape， a previously assumed taxonomysignificant character， also evolved in parallel and convergence frequently. The previous Lagarosolen and Paralagarosolen were characterized by narrow and long （i.e. salverform） corolla tube. However， the present ancestral morphological reconstruction indicated that this salverform corolla evolved at least twice in parallel in Petrocodon from ancestral tubular， and it also evolved into urceolate and tubular quickly in different lineages. The campanulate corolla that characterized Petrocodon s.s. and Wentsaiboea was also suggested to evolve twice convergently within the expanded genus Petrocodon， and this convergence also happened between Petrocodon and Primulina. These suggested that the character of corolla shape （salverform and campanulate） that characterized Lagarosolen， Paralagarosolen， Wentsaiboea and Petrocodon s.s. also evolved homoplasiously in Petrocodon and its allies.

Other characters that were assumed to be characteristic of the previous genera Calcareboea， Dolicholoma and Didymocarpus， whose taxa have been wholly or partially included into Petrocodon， were also suggested to be homoplasious according to the present study. Dolicholoma， a previous monotypic genus， possesses narrow corolla tube like the taxa of the previous Lagarosolen， but differs from the latter by its deep split corolla lobes. However， this character of deep split lobes was also observed in another recently described new species Petrocodon ainsliifolius. According to the previous molecular phylogenetic analysis （Chen et al， 2014）， these two species are distantly related to each other， embedding within lineages with quite different characters， respectively， and thus suggesting convergent evolution of the character （discussed also in Weber et al， 2011）. Calcareboea， a previous monotypic genus， is characteristic of bright red flower and very special corolla structure of four shallowly divided adaxial lobes and one abaxial lobe. Although the bright red flower is unique to this species in Petrocodon and was indicated to be derived from ancestral bluish violet， the red flower which was presumed to be associated with ornithophily （Weber et al， 2011b） is also possessed by some taxa of Aeschynanthus， another distantlyrelated genus of Gesneriaceae. The other characteristic of four adaxial lobes was also clearly indicated to have evolved in parallel within Petrocodon （at least twice independently） and beyond the genus （additionally twice observed leading to Allocheilos and Oreocharis mileensis）. The frequent changes and highly homoplasious evolution of these previously assumed taxonomysignificant floral characters seemed to be the key factors that accounted for the unnaturalness of the previous generic classifications， resulted in taxonomic oversplit of genera.

3.2 Potential synapomorphy that may characterize and differentiate the redefined Petrocodon from its closest relative Primulina

Although the redefined genus forms as monophyletic， separated from its closest relative Primulina， little is known about the synapomorphies that can support the new classification. Particularly， these newly redefined sister genera not only encompass remarkable diversity， but also include morphologically similar species reclassified into Petrocodon and Primulina， respectively. For instance， the two morphologically similar taxa， Pri. tabacum and the previous Pri. guangxiensis （namely Petrocodon guangxiensis） were indicated to be embedded in Primulina and Petrocodon， respectively， according to molecular phylogenetic analysis （Xu et al， 2014）. Also， Wentsaiboea renifolia， Wen. luochenensis and Wen. tiandenensis were once placed in the same genus by very similar gross vegetative and floral appearance， however， the molecular phylogenetic studies clearly suggested the former two species should be placed in Primulina， while the latter was nested within Petrocodon （Wang et al， 2011； Weber et al， 2011b）. These morphologically similar taxa divided into two genera confounded the boundary and brought difficulties to uncover potential diagnostic morphological characters that can distinguish and characterize each of the genera.

After extensive comparisons of Petrocodon with Primulina taxa， we noticed two possible synapomorphic characters that may be used to differentiate these two genera. Firstly， most Primulina taxa have geniculate filaments of the two fertile stamens， with an exception of Pri. tabacum， which is embedded deeply within the genus and possesses almost straight filaments. This character of geniculate filaments is also shared by some other Gesneriaceae lineages like Hemiboea and Lysionotus taxa， therefore it at least can be inferred to be ancestral of the genus Primulina as a whole， though it remains unknown weather this character shared by different Gesneriaceae lineages （genera） is indicative of plesiomorphy or convergent or parallel evolution. In contrast， most Petrocodon taxa we observed have straight or slightly curved filaments of the fertile stamens， being different from the commonly geniculate filaments of Primulina. However， Petrocodon retroflexus we recently published has special filaments， which are geniculate near the base like most Primulina taxa but with an additional loop near the tip where the anthers attached （Guo et al， 2016）. Therefore， this character seemed also to be homoplasious between these two genera though it may differentiate most Petrocodon from Primulina taxa.

Secondly， Primulina taxa usually have obtrapeziformlike stigma （Chiritoidtype stigma）， which is commonly bifid. This type of stigma was assumed to be developed from the lower half of an ancestral biparted stigma， with the upper half reduced （Weber et al， 2011a）. This characteristic of stigma is also shared by some other distantlyrelated lineages， particularly those （e.g. Microchirita， Henckelia， Liebigia， etc.） that were unnaturally placed in the previous genus Chirita （Weber et al， 2011a）. Petrocodon taxa， however， have different stigma types which usually have undivided or bipartitioned capitate stigma（s） [corresponding to the concept of two stigmas in Wang et al （1998）] and may be a derived state in the ancestor of the lineage leading to Petrocodon. The capitatelike versus obtrapeziformlike stigma may be used to distinguish Petrocodon from Primulina.

Anyway， these tentatively proposed characteristics of Petrocodon versus Primulina need to be tested by more future extensive examinations of these characters in these two genera and the outgroups and inferences of their evolutionary trends based on more robust phylogenetic tree with denser sampling of the taxa.

4Conclusions and Prospects

The present study illustrated complicated morphological evolution in the genus Petrocodon， especially for those floral characters that were assumed to be taxonomysignificant. Frequent changes and highly convergent and parallel evolution of these traits in the genus and its close relatives seemed to be key factors that have misled the traditional taxonomy， and brought difficulties to identify possible synapomorphic characters that can be used to support the new classifications based on molecular phylogenetic studies. More extensive investigations should be carried out on both morphologies and molecular phylogenetic studies. On one side， the available phylogenetic studies in Gesneriaceae were mainly based on limited plastid and the nuclear ribosome ITS DNA sequences （Wang et al， 2011； Weber et al， 2011a， b）， which sometimes were likely to yield low resolution or partial or even misleading relationships. So that， denser samplings of DNA sequences from different genomes as well as taxa are required to reconstruct the highly resolved species tree. On the other side， more morphologies including micromorphological and anatomical characters， etc. should be examined and added to the present data set， and their evolutionary trends should be traced on robust phylogenetic framework. More importantly， efforts should also be taken to investigate the underlying causes （e.g. molecular regulatory network from the discipline of evolutionary developmental genetics） and its biological senses （e.g. the fitness adapted to natural selection） of the complicated morphological evolution. All these together would help bridge up the gaps between traditional taxonomy and molecular phylogenetic studies and between phenotypes and genotypes， and advance the understandings of the evolution of this group of typical karst flora and its allies.

5Supporting Information

Table S1 GenBank accessions of the sequences used in the present study.

Fig. S1 Evolutionary trends of four selected flower traits， showing highly homoplasious evolution. The ancestral states of the traits were traced using the maximum parsimony method implemented in Mesquite 2.7.

AcknowledgmentsWe thank Dong Yang and LI Peng Wei from Institute of Botany， Chinese Academy of Sciences for reading the manuscript and providing helpful suggestions. We also send our gratitude to an anonymous reviewer of the manuscript for insightful suggestions.

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